Receptor-mediated uptake of peptides that bind the human transferrin receptor

ABSTRACT

Peptides have been discovered which are capable of binding to and internalizing with the human transferrin receptor (hTfR). The sequences HAIYPRH (Seq. ID No. 1) and THRPPMWSPVWP (Seq. ID No. 2) are capable of binding to and internalizing with the human transferrin receptor. When these molecules were fused with other molecules, the fusion product was internalized in cells expressing hTfR. The sequences have use for targeting other peptides and proteins into cells expressing hTfR.

This application is a continuation of U.S. patent application is acontinuation in part of U.S. patent application Ser. No. 09/995,804filed Nov. 29, 2001, now pending, which takes priority from U.S.Provisional patent application 60/253,940.

This work was supported by USPHS grants R01 HL58339 and IP50 DE/CA 11910and by NCI grant CA-13148. Hence, the United States Government hascertain rights in this invention.

FIELD OF THE INVENTION

This invention relates to use of peptides which target the humantransferrin receptor. Peptides of the invention can be used to directother peptides, proteins and other diagnostic or therapeutic agents intocells for both diagnostic and therapeutic purposes.

BACKGROUND OF THE INVENTION

Previous work relating to redirecting viral vectors in gene therapy byusing short peptide ligands to redirect virus particles to specific celltypes are known. One of the limitations of this strategy is that shortpeptide sequences that bind efficiently to cell surface receptors onspecific cell types must be identified. One experimental approach toidentify such short peptides that holds promise is bacteriophagedisplay.

For more than a decade, phage display has exploited the physical linkagebetween random peptide sequences expressing on phage and the DNAencoding that sequence. This linkage allows for rapid identification ofpeptide ligands. A random peptide sequence is expressed as a fusion witha bacteriophage coat protein and is available for testing as a ligandfor various targets. Phage display has successfully been used toidentify single chain antibodies with specificity for various biologicalmolecules. Phage display strategies can be used to elucidate the aminoacids responsible for protein-protein interactions, to findorgan-specific phage, and to find substrate recognition sequences forenzymes. The process of using multiple rounds of phage display to enrichfor a particular sequence is called biopanning.

The human transferrin receptor (hTfR) has been studied extensively as amodel system for receptor-mediated endocytosis, a marker for cellularproliferation, and a target for therapeutics. The hTfR is ubiquitouslyexpressed and over-expressed at least 100 fold in oral, liver,pancreatic, prostate and other cancers. This increase in transferrinreceptor (TfR) in cancers has been attributed to the increasedmetabolism of these transformed cells, making the hTfR a usefuldiagnostic marker. Because of its expression pattern and pathwaycharacteristics, the hTfR is an attractive target for therapeutics. TheTfR is a dimer composed of two identical 95 kDa subunits and isresponsible for the majority of cellular iron uptake. The type II cellsurface receptor binds 80 kDa transferrin (Tf) and the complex isinternalized through clathrin-coated pits. Iron is released fromtransferrin in the acidic early endosome and the apotransferrin-receptorcomplex is recycled back to the cell surface where apotransferrin isrecycled.

A blast search failed to yield any significant homologies between eitherHAIYPRH (Seq. ID No. 1) or THRPPMWSPVWP (Seq. ID No. 2) to knownproteins, including Tf.

SUMMARY OF THE INVENTION

This invention relates to peptides which are capable of binding to andinternalizing with the human transferrin receptor (hTfR). The sequencesHAIYPRH (Seq. ID No. 1) and THRPPMWSPVWP (Seq. ID No. 2) are capable ofbinding to and internalizing with the human transferrin receptor. Whenthese molecules were fused with other molecules, the fusion product wasinternalized in cells expressing hTfR. The sequences have use fortargeting other peptides and proteins into cells expressing hTfR. Thephage display system using whole cell selective biopanning could also beapplied to find small ligands for other cell surface receptors. Thissequence is not found in human transferrin protein. Furthermore, thissequence does not compete with transferrin itself for binding to thehTfR.

DETAILED DESCRIPTION OF THE INVENTION

It is important that easily produced peptides that can facilitate entryof diagnostically and therapeutically useful peptides and proteins intocells having particular characteristics be available. The identificationof peptides that will facilitate entry of such peptides into cells whichare more likely to be aberrant has particular use. The peptides of theinvention are useful for facilitating entry of diagnostically andtherapeutically useful agents, including peptides and proteins. Sincemalignant cells produce increased expression of hTfR, the peptides,HAIYPRH (Seq. ID No. 1) and THRPPMWSPVWP (Seq. ID No. 2), areparticularly useful for study and treatment of malignancies.

A phage display selection strategy was utilized that resulted inidentification of the peptides. This selection system is based onalternating rounds of negative selection on chicken embryo fibroblast(CEF) cells lacking hTfR and positive selection on chicken embryofibroblast cells expressing hTfR (CEF+hTfR). Biopanning on whole cellswas exploited to select the peptides HAIYPRH (Seq. ID No. 1) andTHRPPMWSPVWP (Seq. ID No. 2). These peptides were able to target amacromolecule to and internalize through the hTfR, as was demonstratedby phage binding, competition and immunofluorescence studies. It wasalso shown that these two peptides bind sites that do not overlap withthe native ligand, transferrin, indicating they could be used in vivofor targeting macromolecules to the endocytic pathway in hTfR-positivecells.

The biopanning procedure could be applied to find small peptide ligandsfor other cell surface receptors. There is a great need to find newepitopes on various cancer cell types for diagnostic purposes. Thesubtractive method of biopanning disclosed herein would be useful forfinding new cell surface markers. Biopanning on whole cells can beespecially useful in situations where the receptor can not be purifiedor does not maintain its native confirmation when isolated.

Materials and Methods:

Cell lines: The two chicken embryo fibroblast cell lines, CEF andCEF+hTfR, used for selective biopanning, were described previously(Collawn, et al, Cell, 63, 1061-1072 (1990) and Odorizzi, et al., J.Cell Biol., 126, 317-330 (1994)). Chicken embryo fibroblasts have beenused extensively for study of hTfR. The native cells express chickentransferrin receptors, but this receptor cannot bind human transferrin.Two cell lines were previously established through stable transductionwith retroviral vectors to yield CEF and CEF+hTfR cells. CEF cells donot express the human transferrin receptor. CEF+hTfR cellsconstitutively express hTfR. Protein expression of hTfR by CEF cells wasperiodically checked by ¹²⁵I-Tf binding. Both cells are grown inmonolayer cultures in Dulbecco's Modified Eagle Medium supplemented with1% chicken serum, 1% bovine calf serum, 1% L-glutamine 200 nM, and 2%tryptose phosphate and maintained at 37° C. in 13% CO₂.

Antibodies: Monoclonal anti-GFP (green fluorescent protein) antibody(Clontech, Palo Alto, Calif.) was used for Western blot analysis andimmunofluorescence at 1:5,000 and 1:250 dilution, respectively. Horseradish peroxidase conjugated goat anti-mouse antibody (Pierce, Rockford,Ill.), Oregon-Green and Texas-Red secondary antibodies (MolecularProbes, Eugene, Oreg.) were used at 1:10,000, 1:250, 1:250 dilution,respectively.

Electrophoretic methods: Samples were dissolved on SDS-PAGE gels by themethods of Laemmli and transferred to nitrocellulose membrane byelectroblotting for Western blot analysis (Laemmli, U. K, Nature, 227,680-685). The membranes were blocked with 5% milk in tris bufferedsaline with 1% Trition X-100 (TBS-TX) (50 mM Tris-HCL, pH 7.5, 0.2 MNaCl, 1% Triton X-100), and incubated with primary antibody in TBS-TXwith 2.5% milk overnight at 4° C. The membranes were then washed inTBS-TX and incubated with peroxidase-conjugated secondary antibody anddeveloped with the enhanced chemiluminescence (ECL) kit in accord withthe manufacturer's instructions (Amersham Pharmacia Biotech,Buckinghamshire, England).

Biopanning: Ph.D.-7™ or Ph.D.-12™ Phage Display Peptide Library Kit (NewEngland Biolabs, Inc, Bevery, Mass.) was used for biopanning on CEF andCEF+hTfR cells. The Ph.D.™ phage display peptide library is based on acombinatorial library of random 7 or 12 amino acid peptides fused to aminor coat protein of the filamentous coliphage M13. In separatestudies, two different phage display peptide libraries were used toselect for 7-mer and 12 mer peptide sequences that could bind the hTfRexpressed on the surface of CEF+hTfR cells. Cells were washed andincubated in serum-free Opti-MEM (Gibco BRL Life Technologies,Gaithersburg, Md.) at 37° C. for 1 hour prior to all biopanningprocedures. Phage binding was carried out at 4° C. in serum-freeOpti-MEM with 1×10⁶ cell/3.5 cm well. Initial biopanning proceduresapplied 2×10¹¹ phage to CEF cells for two hours; unbound phage weretransferred to CEF+hTfR cells for 1 hour. Cells were washed 10 timeswith Opti-MEM, and bound phage was quickly eluted with low pH buffer(0.2M glycine-HCL, pH 2.2) and neutralized with 1M Tris-HCl, pH 9.1.Eluted phage were amplified in 20 ml Luria-Bertani medium (LB)containing E. coli ER2537 (for 7-mer phage) and ER2783 (for 12-merphage) at 37° C. Phage from liquid cultures were obtained by clearingthe supernatant twice by centrifugation at 10,000 rpm for 15 minutes at4° C., and precipitated with ⅙ volume of PEG/NaCl (10% polyethyleneglycol-8000, 2.5M NaCl) at 4° C. overnight. Phage pellets were suspendedin 1 ml TBS (50 mM Tris-HCl, 150 mM NaCl), and precipitated withPEG/NaCl for 1 hour. Amplified phage were resuspended with 200 μl TBS,0.02% NaN₃, and these amplified phage were used for additional rounds ofbiopanning. After each round of biopanning, the final elutes weretitrated, amplified in E. coli, and plated onto LB plates. The plateswere incubated at 37° C. overnight. Individual plaques were subjected toplaque amplification, DNA purification, and DNA sequencing using amodified Sanger sequencing reaction (Sanger, et al., Pro Natl Acad SciUSA, 74, 5463-5467 (1977)) with the appropriate sequencing primers.7-mer sequencing primer: 5′-TGGGATTTTGCTAAAAAC-3′ (Seq. ID No. 5) 12-mersequencing primer: 5′-GTATGGGATTTTGCTAAACAAC-3′ (Seq. ID No. 6)Peptide Synthesis: The peptides HAIYPRH (Seq. ID No. 1), IRHPHYA (Seq.ID No. 3), THRPPMWSPVWP (Seq. ID No. 2), and PWRPSHPVWMPT (Seq. ID No.4) were synthesized on an Applied Biosystems Model 440 by means of thesolid phase peptide synthesis procedure at the Peptide Synthesis CoreFacility of the University of Alabama at Birmingham (UAB) ComprehensiveCancer Center. These peptides were purified by high pressure liquidchromatography, and the molecular weights were confirmed by massspectrometry.Binding and Competition Studies: Purified phage populations wereamplified and were verified to be homogenous through DNA sequencing.Cells were prepared for binding as was described for biopanningprocedures. Preparations of plaque-purified and titered phage (1×10¹¹)were incubated in serum-free Opti-MEM on either CEF or CEF+hTfR cells at4° C. for 1 hour. The cells were washed repeatedly with Opti-MEM andbound phage were eluted with low pH buffer and subsequently titered. Incompetition studies, holo-transferrin (Calbiochem, La Jolla, Calif.) orsynthesized peptides were added to CEF+hTfR cells prior to addition ofthe phage for 1 hour at 4° C. Multiple trials were completed and averagetiters and standard deviations determined. The titers determined onCEF+hTfR cells were divided by the titers determined on CEF cells andmultiplied by 100 to yield fold over control data points.

Modified GFP Constructs: Transferrin from human serum, bovine serumalbumin (BSA), and purified wild-type GFP (wtGFP) were obtained fromSigma (St. Louis, Mo.) and Clontech (Palo Alto, Calif.), respectively.The tagged GFP genes were generated by the PCR with template DNAClontech's GFP vector. The PCR reactions were carried out in a PerkinElmer Cetus DNA Thermal Cycler for 30 cycles of 95° C., 1 minute; 55°C., 1 minute; and 72° C., 1 minute. HAIYPRH (Seq. ID No. 1)-tagged GFPPCR oligonucleotides were: Upstream: (Seq. No. 7)5′-TCTAGATCTGATGAGTAAAGGAGAAGAA-3′ Downstream: (Seq. No. 8)5′-TTAAAGCTTTTAATGGCGCGGATAGATCGCATGTTTGTAGAGCTCAT CCATGCC-3′

THRPPMWSPVWP (Seq. ID No. 2)-tagged GFP PCR oligonucleotides were:Upstream: (Seq. No. 7) 5′-TCTAGATCTGATGAGTAAAGGAGAAGAA-3′ Downstream:(Seq. No. 9) 5′-TAAAGCTTTTACGGCCACACCGGGCTCCACATCGGCGGGCGGTGGGTTTTGTAGAGCTCATCCATGCC-3′The PCR products were purified with the Qiagen Gel Extraction Kit(Qiagen, Valencia, Calif.), and cut with BglII and HindIII restrictionenzymes (Roche, Nutley, N.J.), and subcloned into the pET-32a(+)bacterial expression vector (Novagen, Madison, Wis.). The resultingexpression vector was verified using a modified Sanger sequencingmethod. The tagged GFP expression plasmids were transformed intoBL21/DE3 E. coli and expression was induced for 3-4 hours with 1 mMisopropyl-β-D-thioglactopyranoside (IPTG) when the culture O.D,₆₀₀=0.5.Cells were pelleted, then resuspended in phosphate buffer with 20 mMimidazole followed by passage through a French press at 10,000-15,000psi. Cell lysates were passed over a PisTrap nickel column (AmershamPharmacia Biotech, Piscataway, N.Y.). The column was washed and finallyeluted using an imidazole gradient. The purified protein was assayed bySDS-PAGE followed by Coomassie staining and Western blot analysis with aGFP monoclonal antibody (Clontech). ECL development was carried out asper the manufacturer's instructions (Amersham Pharmacia Biotech).Protein concentrations were determined with the Bio-Rad Protein Assaykit (Bio-Rad Laboratories, Hercules, Calif.).Immunofluorescence: CEF+hTfR cells were grown on glass coverslips to 50%to 75% confluence. The coverslips were washed and incubated inserum-free Opti-MEM media at 37° C. for 1 hour. Then 2 μg of wild-typeGFP (Clontech), HAIYPRH (Seq. ID No. 1)-tagged GFP, THRPPMWSPVWP (Seq.ID No. 2)-tagged GFP, or Texas-Red Tf (Molecular Probes) was applied tocells in serum-free Opti-MEM media for 1 hour at 4° C. or 37° C. Cellswere washed with Opti-MEM, then fixed in 3% formaldehyde for 30 minutesat 4° C. Alternatively, the cells were acid-washed with 0.2 Mglycine-HCl, Ph 2.2, prior to fixation. A GFP monoclonal antibody(Clontech) was used in conjunction with an Oregon-Green Goat Anti-Mouse(Molecular Probes) to augment GFP fluorescence. All slides werecounterstained with DAPI (2(4Amidinophenyl)-6indolecarbamidinedihydrochloride) (Sigma). The microscopic slides were mountedin Prolong™ antifade medium (Molecular Probes). Images were captured onan AX70 microscope with Olympus Camera (Olympus, Melville, N.Y.) andanalyzed with ESPRIT software (Life Science Resources, Cambridge,England). Final figures were assembled using Microsoft Power Point(Microsoft Corp., Redmond, Wash.). For colocalization studies, CEF+hTfRwere incubated with 2 μg/ml GFP fusion protein and 2 μg/ml of Texas-RedTf for 1 hour and processed as described above.Standard Analysis: Purified proteins (transferrin, wtGFP, GFP-HAIYPRH(Seq. ID No. 1) and GFP-THRPPMWSPVWP (Seq. ID No. 2) were labelled with¹²⁵I to a specific activity of 1-2 μCi/μg with CPM/μg determined by agamma counter and Bradford assay. CEF+hTfR cells were plated induplicate at a density of 7.5×10⁴ cells/well in 24 well dishes and grownovernight. Cells were washed and incubated in serum-free Opti-MEM for 1hour at 37° C. Cells were placed on ice with the various amounts oflabelled protein in a total of 200 μl of cold 0.1% BSP in phosphatebuffered saline (PBS). After 1 hour, the unbound protein was removed andcells were washed 4 times with 0.1% BSA in PBS. 1 M NaOH was added tolyse the cells for determining the bound fraction. Both unbound andbound fractions were counted in a gamma counter and binding affinitieswere determined using Scatchard analysis. Studies were repeated 3 timesand yielded comparable binding affinities for all proteins tested.

EXAMPLE 1

The biopanning procedure with chicken embryo fibroblast cells wasperformed as described above. The procedure used both negative andpositive binding steps to isolate specific peptide sequences that bindthe hTfR.

After the cells were incubated in serum-free media Opti-MEM at 37° C.for 1 hour to remove Tf found in the serum, ice-cold serum-free Opti-MEMmedia was applied and the cells were held at 4° C. throughout theselected process to prevent internalization of the receptor. Theoriginal phage library containing 2×10¹¹ phage was applied to CEF cellsfor 2 hours. Unbound phage were transferred to another well of CEF cellsfor an additional hour, before transferring the unbound phage to a wellof CEF+hTfR cells. After extensive washing, the bound phage were removedwith low pH buffer and subsequently neutralized. The eluted phage weretitered and amplified in E. coli. After each amplification step multipleplaques were selected for sequencing. The amplified eluted phage wereapplied to CEF cells to begin the biopanning process again. This cyclewas carried out 10 rounds for the 7-mer peptide library and 7 rounds forthe 12-mer peptide library to achieve significant enrichment of a singlesequence above all others. Sequencing of individual phage plaquesallowed for the monitoring of sequence convergence during multiplerounds of biopanning. Phage titers of total phage eluted were determinedand were noticed to increase after each round of biopanning. The mostprominent sequence selected from the 7-mer library was HAIYPRH (Seq. IDNo. 1) (7-mer) while the 12-mer library converged to the sequence ofTHRPPMWSPVWP (Seq. ID No. 2) (12-mer). There were no other sequencesthat arose consistently throughout the biopanning procedure.

Phage that did not bind the CEF cells were applied to CEF+hTfR cells andthe bound phage were eluted with low pH buffer. The eluted phage wereamplified for additional rounds of biopanning. Between each round, thephage were titered and sequenced to monitor convergence of sequence.

EXAMPLE 2

As an initial test to determine whether the isolated phage bound tohTfR, a phage binding study was performed. Homogeneous pools of fivedifferent isolates from the 7-mer phage and five isolates from the12-mer phage were each amplified, purified and verified by DNAsequencing. Individually, 109 phage were applied to CEF or CEF+hTfRcells. Phage were bound to CEF or CEF+hTfR cells for 1 hour at 4° C.,then washed extensively with Opti-MEM to remove unbound phage. Boundphage were eluted with low pH buffer, neutralized, and titered on a lawnof E. coli. Titering each phage on both cell types was repeated threetimes, and average titers and standard deviations were determined.Considering the 7-mer sequences, it was found that significantly highertiters were obtained only with HAIYPRH (Seq. ID No. 1) phage bound toCEF+hTfR cells when compared to other phage tested. In studies relatingto the 12-mer sequences, it was found that the THRPPMWSPVWP (Seq. ID No.2) phage had higher titers on CEF+hTfR cells than the other 12 aminoacid phage tested. On CEF cells, which do not express hTfR, all phagetested bound at the same low efficiency. A low level of non-hTfRdependent binding is expected, due to interactions between phage coatproteins and the various proteins on the surface of chicken embryofibroblast cells. Titering studies demonstrated that phage containingeither peptide sequence HAIYPRH (Seq. ID No. 1) or THRPPMWSPVWP (Seq. IDNo. 2) bound CEF+hTfR cells more efficiently than any other phage testedand that this higher binding depends on the presence of humantransferrin receptor.

EXAMPLE 3

Competition studies were conducted to determine whether the two phagesbound the same region of the hTfR as serum Tf itself. It was found thatthe titers of HAIYPRH (Seq. ID No. 1) or THRPPMWSPVWP (Seq. ID No. 2)phage bound to CEF+hTfR cells in the presence of various added peptidesor Tf were significant. The HAIYPRH (Seq. ID No. 1) phage was competedaway to background levels only by the HAIYPRH (Seq. ID No. 1) peptideand not by the scrambled sequence of IRHPHYA (Seq. ID No. 3). The 12-merTHRPPMWSPVWP (Seq. ID No. 2) phage was only competed by the THRPPMWSPVWP(Seq. ID No. 2) peptide and not by the scrambled 12-mer sequencePWRPSHPVWMPT (Seq. ID No. 4). Other peptides tested failed to competeaway the phage from binding the cells. Interestingly, the binding ofeither phage was unaffected by the presence of Tf, suggesting that eachphage sequence has a different binding site on the hTfR. Due to asynthesis error, a peptide with the sequence HAIYPNH (Seq. ID No.14) wasalso synthesized. Competition studies were completed with this peptidewhich disclosed no effect on the HAIYPRH (Seq. ID No. 1) phage binding.The result suggests that the 7-mer phage binding depended on thearginine in the original HAIYPRH (Seq. ID No. 1).

EXAMPLE 4

To evaluate the sufficiency of capacity of the peptides to mediateuptake of carrier protein, GFP fusion proteins were prepared.Immunofluorescence was used to determine if the GFP-peptides fusionconstructs were internalized using the following assay. GFP-peptideconstructs were cloned with a C-terminal peptide addition of eitherHAIYPRH (Seq. ID No. 1) (GFP-HAIYPRH) or THRPPMWSPVWP (Seq. ID No. 2)(GFP-THRPPMWSPVWP). These constructs were expressed and purified togreater than 95% by Coomassie staining. Purified proteins were appliedto CEF+hTfR cells at 4° C. (which prevents endocytosis) or at 37° C.Cells were washed with Opti-MEM, fixed and processed as described underthe Materials and Methods section above. Alternatively, the cells werewashed with low pH buffer prior to fixation. This acid wash determinedwhether the protein was endocytosed by removing proteins bound at thecell surface.

Immunofluorescence microscopy was used to follow binding andinternalization of the wtGFP, GFP fusion proteins and transferrin toCEF+hTfR cells. Wild-type GFP was used as a negative control, while Tfconjugated to the Texas-Red fluorochrome was used as a positive control.The conjugation of Texas-Red to Tf has been shown previously not todiminish interaction with the hTfR. In all studies, cell nuclei werecounterstained with DAPI.

Immunofluorescence images of the localization of various proteinsapplied to CEF+hTfR cells at either 4° C. or 370 were studied. At 4° C.,endocytosis was blocked so that all proteins remain at the cell surface,and an acid wash removes all cell surface bound proteins. Whenimmunofluorescence of the various proteins was studied on CEF+hTfR cellswhich had been incubated at 37° C. for one hour, localization ofGFP-HAIYPRH (Seq. ID No. 1), GFP-THRPPMWSPVWP (Seq. ID No. 2) orTexas-Red Tf was found on cells that had not been exposed to acid wash.The total fluorescence shown could result from both cell surface andendocytosed proteins. There was minimal binding of wtGFP even without anacid wash.

The cells that had undergone a low pH buffer wash to enableidentification of proteins that had been endocytosed were evaluated.While wtGFP was unable to be endocytosed into CEF+hTfR cells, bothGFP-HAIYPRH (Seq. ID No. 1) and GFP-THRPPMWSPVWP (Sea. ID No. 2) showeda speckled pattern of fluorescence typical of endocytosed ligands. TheTexas-Red Tf was readily endocytosed into the CEF+hTfR cells andproduced a spotted pattern similar to that seen with the two GFP fusionproteins.

In separate studies at 4° C. or 37° C., CEF cells were used forimmunofluorescence binding assays and neither of the GFP fusion proteinsor transferrin bound or internalized these cells, as was expected, sincethese CEF cells lack the hTfR. Immunofluorescent internalization studieswere also performed with Hela cells and yielded identical result toCEF+hTfR cells.

EXAMPLE 5

The phage titering experiments demonstrated that neither peptidesequence competed with Tf or hTfR binding. Co-localization studies wereconducted with both GFP-Peptide and Texas-Red Tf constructs. Cells wereincubated at 37° C. for 1 hour with Texas-Red Tf and either GFP-HAIYPRH(Seq. ID No. 1) or GFP-THRPPMWSPVWP (Seq. ID No. 2). Cells were acidwashed immediately, fixed and stained with DAPI. Images were capturedusing the appropriate filter and overlaid with images captured with theDAPI filter. Merging GFP, Texas-Red Tf and DAPI images yielded theco-localization images. The fluorescent patterns of the GFP fusionproteins and Tf were identical after acid wash. This result indicatedthat the GFP-peptides were internalized and bound in the sameintracellular compartment as Tf.

EXAMPLE 6

Purified transferrin, wtGFP, GFP-HAIYPRH (Seq. ID No. 1) andGFP-THRPPMWSPVWP (Seq. ID No. 2) were labelled with ¹²⁵I on tyrosineresidues to a specific activity of 1-2 μCi/μg. Serial dilutions oflabelled proteins were incubated with CEF+hTfR cells on ice inPBS-0.1%BSA in duplicate wells. After 1 hour, the unbound fraction wasremoved and cells were washed four times. Cells were removed from thewell with 1N NaOH. The unbound and bound fractions were counted in agamma counter and fmoles of bound and unbound were calculated. Scatchardplots were derived by plotting bound versus bound/free of an averagevalue generated by the duplicate wells. A best of fit line was generatedusing the Excel program (Microsoft Corp.) and the binding affinitieswere determined by the slope of the plotted lines. Repetitive trialsproduced comparable binding affinities. The affinity of Tf was found tobe 2.7×10⁻⁹, similar to previous reports. The affinity for wtGFP andGFP-HAIYPRH (Seq. ID No. 1) were determined to be nominal at 2.4×10⁻⁴Mand 3.6×10⁻⁴M, respectively. This low affinity of GFP-HAIYPRH (Seq. IDNo. 1) was attributed to the ¹²⁵I labelling of the tyrosine residue inthe peptide, which could block this peptide's interaction with the hTfR.However, GFP-THRPPMWSPVWP (Seq. ID No. 2) was shown to have 2.3×10⁻⁸Maffinity for CEF+hTfR cells, indicating that its affinity was only10-fold lower than the native Tf ligand.

Peptides containing the sequences HAIYPRH (Seq. ID No. 1) andTHRPPMWSPVWP (Seq. ID No. 2) can be used to target viral vectors, aswell as proteins, to the endocytic pathway via the hTfR. Competitionstudies suggest that transferrin, the 7-mer sequence and the 12-mersequence all bind unique sites on the hTfR, since they each failed tosignificantly compete with each other for hTfR binding. This findingsuggests further advantages for use of these peptides for transductionof therapeutic ligands, since there is no disruption of transferrin'sdelivery of iron to cells.

Due to the characteristics and expression pattern of the hTfR, ligandsspecific for this receptor may be used as targeting agents with antigenas well as diagnostic agents such as imaging agents or radioisotopes. Ithas been shown that early endosomes are essential for the properendocytosis, sorting and presentation of antigen by majorhistocompatibility class II. The targeting of antigens to the hTfRenhances antigen entry into the endocytic pathway and boosts antigenpresentation.

It is possible to conjugate the peptides of the invention to liposomesor viral vectors containing active agents such as chemotherapeutics.(See Eavarone, et al, “Targeted Drug Delivery to C6 Glioma byTransferrin-coupled Liposomes”, Proceedings of the World BiomaterialsCongress 2000, (John Wiley and Sons, Inc.) (2000)). Alternatively,chemotherapeutics may be conjugated directly with the peptides of theinvention for targeting agents to transferrin receptor-rich cells.Because the peptides of the invention do not interfere with binding ofhuman transferrin to the hTfR, different agents may be administeredwherein one conjugate targets the hTfR uses transferrin as the targetingagent and another conjugate targets the hTfR using a peptide of theinvention as a targeting agent.

EXAMPLE 7

Transferrin receptor binding peptide sequences to adenovirus proteins inaccord with the teachings of U.S. Pat. No. 6,312,699, which isincorporated herein by reference in its entirety. As described inexample 2 of U.S. Pat. No. 6,312,699, short peptide ligands such asHAIYPRH (Seq. ID No. 1) and THRPPMWSPVWP (Seq. ID No. 2) are fused ontothe carboxyl-terminus of the adenovirus fiber protein. Oligonucleotidesencoding these amino acid sequences are designed and synthesized andannealed together for cloning into the unique BamHI restrictionendonuclease cleavage site in plasmid pTKgpt-3S (cited in example 2 ofU.S. Pat. No. 6,312,699). Examples of such oligonucleotides are:

For HAIYPRH (Seq. ID No. 1): Sense: (Seq. ID No. 10) 5′ GA TCC CAT GCGATC TAT CCG CGC CAT TAA 3′ Antisense: (Seq. ID No. 11) 5′ G ATC TTA ATGGCG CGG ATA GAT CGC ATG G 3′

For THRPPMWSPVWP (Seq. ID No. 2): Sense: (Seq. ID No. 12) 5′ GA TCC ACCCAC CGC CCG CCG ATG TGG AGC CCG GTG TGG CCG TAA 3′ Antisense: (Seq. IDNo. 13) 5′ G ATC TTA CGG CCA CAC CGG GCT CCA CAT CGG CGG GCG GTG GGT G3′These oligonucleotides are designed with BamHI cohesive ends that can becloned into the BamHI cleavage site developed in Example 2 of U.S. Pat.No. 6,312,699. The specific amino acid sequence added to fiber inExample 2 was designed to extend the new transferrin receptor-bindingligand away from the bulk of the fiber protein, increasing itsaccessibility to the new receptor molecule. The fiber protein, modifiedto include a linker and a ligand, could still form a trimer.

The non-viral ligands can be attached to the carboxyl terminus of thefiber protein via a peptide linker by expression of a geneticallyengineered nucleic acid sequence encoding the fiber protein, linker, andligand. Alternatively, one could use PCR mutagenesis to introduce thesetwo sequences into plasmid pTKgpt-3S, using synthetic oligonucleotidesas in example 4 of the cited patent.

EXAMPLE 8

The Tf receptor binding peptides can be used to enhance antigen deliveryin antigen-presenting cells. These peptide sequences are applied toincrease the potency of vaccines, since antigen-presenting cells oftentake up the antigens contained in vaccines poorly. To enhance antigendelivery and, therefore, antibody and cytotoxic T cell responses, thesepeptides are chemically coupled to the antigen of interest or preparedas a recombinant protein that contains these Tf receptor-bindingpeptides. For preparation of the recombinant antigen containing the Tfreceptor binding peptide, coupling is accomplished using standardrecombinant DNA techniques as in other examples provided (for example,fusions of HAIYPRH (Seq. ID No. 1) and THRPPMWSPVWP (Seq. ID No. 2) toGFP or adenovirus fiber proteins.) The recombinant proteins can beexpressed in any number of protein expression systems includingbacterial, baculoviral, and mammalian expression systems.

For chemical conjugation of the Tf receptor binding peptides, thepeptides are coupled using chemical crosslinkers such as succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC; Piece Chem. Co.,Rockford, Ill.). Obviously, any chemical crosslinker could be used forthis purpose. In our applications, we have coupled 10 mg of antigen to a5 to 30-fold molar excess of SMCC in 50 mM Hepes buffer (pH 7.4) for 1 hat room temperature.

EXAMPLE 9

SMCC-modified antigen is purified by gel filtration to remove theunbound crosslinker. Using this particular crosslinker, peptides areprepared with an amino-terminal linker sequence with a cysteine residuefollowed by a nonspecific linker sequence (glycine-proline-glycine) tofacilitate the coupling reaction. (The leader sequence can changedepending on the nature of the crosslinker.) After the leader sequence,the 7- or 12-residue Tf receptor binding peptide is attached. Thepeptides are added to SMCC-modified antigens at same molar ratio as isused with the cross-linker. The reactions are incubated overnight atroom temperature.

Reaction products are separated by gel filtration and the number ofcross-linkers and/or peptides coupled to the antigen is determined byMALDI-TOF mass spectrometry. These Tf receptor binding peptide-modifiedantigens can then be used as a vaccine using standard vaccinationprotocols.

The advantage of the peptide-coupled antigens is that substantially lessantigen will be required for inducing antibody-based responses. Since anumber of peptides can be coupled to each antigen molecule, antigenicresponses should be dramatically enhanced.

EXAMPLE 10

The peptides of the invention may also be coupled with chemotherapeuticagents. Using 2 equivalents of either peptide HAIYPRH (Seq. ID No. 1) orTHRPPMWSPVWP (Seq. ID No. 2) or a combination of the two, to oneequivalent of methotrexate the peptides of the invention are coupled tomethotrexate using the methods of examples 8 and 9. The resultingproduct is formulated in buffered saline and administered to the patientin sufficient amount to provide a concentration of 0.3 to 5 μM in theserum when administred intravenously.

EXAMPLE 11

The methotrexate bound to the peptides of the invention is prepared asin example 10. However, the methotrexate bound to the peptides is thenformulated in liposomal form for intravenous administration. Liposomalcompositions may also be administred by mouth or directly to theaffected tissue.

Examples of other antineoplastic agents that might be conjugated to thepeptides of the invention, either directly or through conjugation to orincorporation in liposomes containing the sequences of the invention,such liposomes containing antineoplastic agents which may be bound tothe peptides of the invention, to target cells rich in human transferrinreceptors include (but are not limited to) cisplatin, nitrogen mustards(including chlorambucil), ethylenimines, methylmelamines, nitrosoureas(including carmustin, lomustine, etc.) and doxorubicin. Theantineoplastic agents would be administered in accord with the methodsusually used for the particular agent and disease. However, because ofthe selective targetting of the agent by the peptides, lower dosage isrequired. (The lowering of dosage of the antineoplastic agent can be asmuch as 80%.) Furthermore, because the over-all dosage of the neoplasticagent can be decreased, the active agent can be administered for alonger period of time and more frequently than when the non-targettingagent is employed.

The compositions with the peptides bound to antigens or antineoplasticagents may be administered directly to the involved tissues. Forexample, in cases of maligancy of the respiratory tract, the agents maybe administred by inhalation. In treating maligancies of the brain orspinal cord, the agents may be administered intrathecally. For oraladministration, the peptide-bound agents may be administred in entericcoated dosage forms to prevent destruction in the stomach.

1. A composition of matter comprising a purified peptide containing thesequence which is HAIYPRH (Seq. ID No. 1).
 2. A composition comprisingat least one peptide containing within its sequence at least onesequence which is HAIYPRH (Seq. ID No. 1), wherein said peptide is fusedto a protein or another peptide.
 3. A composition of claim 2 whereinsaid sequence containing at least one sequence HAIYPRH (Seq. ID No. 1)is fused to a chemotherapeutic agent.
 4. A composition of claim 2wherein said sequence containing at least one sequence which is HAIYPRH(Seq. ID No. 1) is fused to an imaging agent.
 5. A composition of claim4 wherein said sequence containing at the sequence HAIYPRH (Seq. IDNo. 1) is fused to a fluorescing agent.
 6. A composition of claim 2wherein said sequence containing the sequence HAIYPRH (Seq. ID No. 1) isfused to an antigen.
 7. A composition of matter comprising a constructcontaining a DNA sequence which encodes at least one of the peptide ofthe formula HAIYPRH (Seq. ID No. 1) which is also bound to a nucleotidesequence which encodes a peptide other than HAIYPRH (Seq. ID No. 1).